We investigated whether DNA polymerase beta activity and expression in rat adrenal glands and testes are controlled by the cAMP dependent protein kinase (A-kinase) phosphorylation system in addition to anterior pituitary hormones. DNA polymerase beta mRNA expression in rat testes was decreased by hypophysectomy and recovered with administration of gonadotropic hormone, suggesting that this enzyme is controlled at the mRNA level by this pituitary hormone. In addition, DNA polymerase beta activity in the adrenal glands and testes and the amount of mRNA in the testes increased when cAMP was administered to the normal rat. This activity was decreased by administration of the cyclic AMP-dependent protein kinase inhibitor, H(8). Moreover, when alkaline phosphatase was added to the assay system in vitro, a decrease in DNA polymerase beta activity was observed. These findings indicate that changes in the activity and expression of DNA polymerase beta are mediated via cAMP and the A-kinase system, and that phosphorylation of this enzyme is also involved in this expression.